Copending application Ser. No. 898,874 filed Aug. 20, 1986 and entitled "Biologically Active Material Test", now U.S. Pat. No. 4,716,123, discloses a technique for measuring concentration of antigen molecules in a test sample. A multiplicity of spherical latex particles and a dipstick or slide, both of which are surface-pretreated to be biologically active, are placed in a solution containing the test sample. Antibodies on the surfaces of the dipstick and particles react with antigens in the solution and bond the particles to the surface of the dipstick. The concentration or density of particles on the surface of the dipstick, measured as the number of particles per unit area, is thus a measure of antigen concentration in the test solution. Time required to complete a test is a function, in part, of rate of diffusion of particles in solution, which in turn is a function of particle size. Ability to measure concentration also varies with particle size. In a compromise between test speed and measurability, a particle size of less than 1 .mu.m, and particularly in the range of 300 to 500 nm, is presently preferred. For purposes of calibrating the test procedure, it is also preferable that the test particles have an essentially monodisperse size distribution--i.e., of preselected uniform size .+-.10%.
According to conventional practice, the density of particles attached to the surface of the capture strip or dipstick is determined by visual inspection by a trained operator, and a subjective score ranging from 0 for a blank strip to 5 for a full strip is assigned to the test strip depending upon observed particle density. For a well-trained technician, substantial correspondence can be obtained between such visual assignments, termed visual immuno-assay (VIA) scores, and actual particle density. Indeed, the subjective VIA score assigned by a well-trained technician correlates substantially linearly with the logarithm of particle density. However, it will be apparent that accuracy of quantitative analysis is strongly dependent upon training and consistency among clinical personnel.
It has also been proposed to measure particle surface concentration employing an optical microscope. However, because particle size is on the same order of magnitude as the wavelengths of visible light (about 400 to 700 nm), and because the particle distribution is often non-uniform, particle counts must be taken over a large enough area of the sample surface to allow statistical averaging to be valid. To perform visual counts over such an area is tedious and time-consuming. Furthermore, the presence of scratches, dust and other imperfections on or in the dipstick, on the same order of magnitude as particle size, contributes to inaccuracy of this method.
It is an object of the present invention to provide a measurement technique which yields an accurate indication of particle concentration, which is inexpensive to implement in immunodiagnostic assays, which yields rapid determination of particle concentration, and which may be readily implemented by relatively unskilled clinical personnel. Another object of the invention is to provide a measurement technique of the described character which provides a digital measure of particle concentration in the range of 0 to 5 and is thus correlated with the manual VIA test score method previously described.
A more general object of the invention is to provide an accurate and inexpensive method of optically measuring concentration of particles on a medium or carrier having particle sizes on the same order of magnitude as the measurement light wavelength, and to provide apparatus for implementing such method.